1. Field of the Invention
The present invention relates to transgenic bioluminescent plants. More specifically, the present invention relates to plants, cells of which have been transfected via Agrobacterium or other means known to those in the art, with nucleic acid molecules encoding luciferase and luciferin such that the resulting plant luminesces, in whole or in part. The transfected nucleic acid molecules may be operably linked to, and their expression regulated by, promoters in order to control the incidence, timing and duration of the genetically engineered bioluminescence. Bioluminescent emissions may further be transformed such that light of varying wavelengths is emitted from the plants.
2. Prior Art
It has been known in the art for some time that a certain class of enzymes, known as luciferases, will bioluminesce in the presence of compatible substrates, referred to generally as luciferins. Luciferases are a broad class of proteins that exhibit little homology and are found in terrestrial and marine bacteria, jellyfish, fireflies and a variety of other organisms. Nucleic acid molecules which encode luciferase have been identified, and their bioluminescent activities have been used extensively to study gene regulation and expression. By inserting luciferase protein encoding sequences downstream from a promoter to be studied, one may easily tell when that promoter has been activated by the resulting bioluminescence.
Luciferins, the substrates for luciferases, tend to be complex organic molecules. Bacterial luciferins, such as those found in Vibrio, Photobacterium and Xenorhabdus, have been described as a complex of fatty acid reductase proteins. Some luciferins are thought to be formed by means of complex catabolic pathways. Others, such as the jellyfish luciferin coelenterazine, result from the cyclization of amino acids of a polypeptide. Until recently, nucleic acid molecules encoding the luciferin complex were not known. This meant that in order to detect luciferase, luciferin had to be applied directly to organisms expressing luciferase. The luciferin had to be absorbed by the target, and as a result, suitable hosts were generally limited to cells, relatively thin tissue cultures, and very small seedlings. Organisms or cells expressing luciferase were lysed and exposed to a luciferin solution. This obviously killed the host organism.
There has been a significant amount of work done to improve the use of luciferase in studying gene expression; however, all efforts have been limited by the inability to produce in vivo bioluminescence without the addition of chemicals, outside of a laboratory environment, and in larger organisms.
U.S. Pat. No. 5,093,240 to Inouye et al., incorporated by reference, discloses the transgenic use of the jellyfish luciferase known as aequorin and derivatives thereof. This patent also discloses the use of a luciferase enzyme in a vector designed for mass production. The patent suggests that large quantities of luciferase may be grown in bacterial culture. The patent does not disclose nucleic acid molecules which produce intracellular luciferin. Nor does it contemplate or disclose suitable methods for inserting sequences that encode luciferin into a plant cell.
U.S. Pat. No. 5,162,227 to Cormier, incorporated by reference, also discloses recombinant DNA vectors into which a sequence encoding luciferase has been inserted. As with the above referenced patent, it contemplates use of these vectors for mass production of luciferase in bacterial culture. It further contemplates use of the luciferase gene as a marker or selection gene sequence. It does not, however, contemplate the insertion of a luciferin coding sequence into the vector, in vivo bioluminescence, or the formation of a vector suitable for transfection of plant cells.
U.S. Pat. No. 5,422,266 to Cormier et al., incorporated by reference, discloses an invention very similar to the one described in the above paragraph. It discloses the insertion of a luciferase gene into a vector suitable for use in microorganisms. Like the above mentioned patent, it contemplates neither the additional insertion of a luciferin coding sequence, in vivo bioluminescence, nor the use of vectors suitable for insertion into plant cells.
U.S. Pat. No. 5,583,024 to McElroy et al., incorporated by reference, discloses use of a second luciferase that is useful in transcription assays. This patent contemplates using the luciferase to quantify transcription of various promoter sequences. It requires lysis, and thus death, of the transformed cells. It does not contemplate either in vivo bioluminescence or the use of a luciferin encoding sequence.
U.S. Pat. No. 5,976,796 to Szalay et al., incorporated by reference, discloses a fusion protein comprising a luciferase and a fluorescing protein. This patent contemplates the use of the fusion protein as a double marker in transcription assays. It does not provide for intracellular luciferin or in vivo bioluminescence.
U.S. Pat. No. 5,221,623 to Legocki et al., incorporated by reference, discloses the use of the lux bacterial luciferase gene in transcription assays of various promoters. It does not contemplate in vivo bioluminescence in mature plants or the use of a luciferin encoding sequence. Furthermore, the lux bioluminescence mechanism requires a substantial concentration of organic aldehydes, and this patent discloses applying aldehyde vapors to the microorganisms. This would be impractical for use in the present invention.
U.S. Pat. Nos. 5,876,995 and 6,247,995 B1 to Bryan, both incorporated by reference, disclose the use of bioluminescent luciferase/luciferin mechanisms for use in a wide variety of novelty items. These patents do not disclose recombinant uses for luciferase/luciferin recombinant DNA. Nevertheless, the specifications of these patents are very useful in that they give a very detailed, textbook-like description of the entire field of bioluminescence.
U.S. Pat. No. 5,741,668 to Ward et al., incorporated by reference, discloses a polypeptide capable of spontaneously forming, in vivo, the luciferin coelenterazine. This patent further discloses the mass production of coelenterazine by expressing appropriate coding sequences in bacterial cultures and harvesting the resulting proteins. It does not contemplate combining luciferin coding sequences with those for luciferase in a single vector and using that vector to form bioluminescent organisms such as mature plants.
It is therefore desirable to provide a method for causing bioluminescence in a mature multicellular organism, such as a plant.
It is also desirable to provide a method for inducing bioluminescence without the need to apply chemicals to an organism.
It is also desirable to provide for a mature plant capable of bioluminescence outside of a laboratory setting and without the need of applying special chemicals.
It is also desirable to provide a mature plant capable of bioluminescence where the timing of that bioluminescence is controlled, or when such bioluminescence can communicate important information about the relative health or condition of the plant.
It is also desirable to provide a mature plant capable of bioluminescence where the bioluminescent emission can be transformed and the wavelength of the light emitted from the plant can vary.